CN115868478A - Application of trehalose and betaine in frozen cells - Google Patents
Application of trehalose and betaine in frozen cells Download PDFInfo
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- CN115868478A CN115868478A CN202211631298.7A CN202211631298A CN115868478A CN 115868478 A CN115868478 A CN 115868478A CN 202211631298 A CN202211631298 A CN 202211631298A CN 115868478 A CN115868478 A CN 115868478A
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- HDTRYLNUVZCQOY-UHFFFAOYSA-N α-D-glucopyranosyl-α-D-glucopyranoside Natural products OC1C(O)C(O)C(CO)OC1OC1C(O)C(O)C(O)C(CO)O1 HDTRYLNUVZCQOY-UHFFFAOYSA-N 0.000 title claims abstract description 37
- HDTRYLNUVZCQOY-LIZSDCNHSA-N alpha,alpha-trehalose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-LIZSDCNHSA-N 0.000 title claims abstract description 37
- KWIUHFFTVRNATP-UHFFFAOYSA-N Betaine Natural products C[N+](C)(C)CC([O-])=O KWIUHFFTVRNATP-UHFFFAOYSA-N 0.000 title claims abstract description 36
- KWIUHFFTVRNATP-UHFFFAOYSA-O N,N,N-trimethylglycinium Chemical compound C[N+](C)(C)CC(O)=O KWIUHFFTVRNATP-UHFFFAOYSA-O 0.000 title claims abstract description 36
- 229960003237 betaine Drugs 0.000 title claims abstract description 36
- HDTRYLNUVZCQOY-WSWWMNSNSA-N Trehalose Natural products O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-WSWWMNSNSA-N 0.000 title claims abstract description 34
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- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 10
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Images
Abstract
The invention relates to the field of cell cryopreservation, and discloses application of trehalose and betaine to cryopreservation of cells. The cell preparation comprises cells, trehalose and betaine, wherein the weight ratio of the trehalose to the betaine is 1-4.2. The invention can realize the long-term preservation of cells (particularly mesenchymal stem cells), can be directly used without the complicated steps of recovery culture amplification, washing and the like, saves the time for recovering the cultured cells and an operation platform required by cell operation, and is beneficial to saving equipment and labor cost.
Description
Technical Field
The invention relates to the field of cell preparations, in particular to application of trehalose and betaine in cryopreservation of cells.
Background
Mesenchymal Stem Cells (MSCs) are a class of cell populations with self-renewing, multi-directional differentiation potential. MSCs have the characteristics of low immunogenicity, multiple origins, multiple differentiation potential, strong paracrine function, etc., and can restore the homeostasis of the organism through systemic functional regulation in the organism. Various studies have been conducted to investigate the therapeutic effects of MSCs in diseases, including treatment of myocardial infarction, autoimmune diseases, diabetes, liver injury, kidney injury, and the like. MSCs treatment involves a variety of approaches including anti-inflammatory, anti-oxidative stress, anti-endoplasmic reticulum stress, activation of the immune system, and promotion of regeneration at injured sites.
Currently, methods of application of MSCs include intravenous infusion, topical injection, and the like. Intravenous infusion is the most common method due to its advantages such as simple operation and strong systemic regulation. Currently in research and use, a course of MSCs usually requires multiple infusions, each at intervals, for a course of about 1-2 months. At present, the fractionated infusion of the MSCs is complicated to operate, the recovery passage and the amplification of the MSCs need to be carried out before each infusion, and a single flow usually needs 3-5 days, generally 3-5 times. Secondly, the preservation of MSCs, culture conditions and operator variability also affect the efficacy of MSCs therapy. In addition, the greatest obstacles to the use of MSCs therapy for some acute and rapid onset diseases in pet clinics include long MSCs preparation time, high handling requirements, short storage time, and the like.
MSCs have long been limited in the time and platform of manipulation for large-scale application in disease treatment. Some of the research has focused on the storage temperature and environment of MSCs. Studies by G a lvez-Mart i.e. showed that MSCs stored at 4 ℃ in short haul (< 60 hours) had greater cell viability than at 25 ℃ and 37 ℃. Gniadek et al added 5% human serum albumin to MSCs preparations increased the cryopreservation efficiency of MSCs. Horcharoensuk P et al added proline as a protective agent on 0.9% physiological saline to enhance short haul transport efficiency of MSCs. In short-term transport (less than or equal to 60 hours) of MSCs, 0.9% physiological saline, phosphate buffer solution, ringer's solution, etc. are often used as a vehicle, but the above-described scheme still does not satisfy the large-scale preparation and long-term preservation of MSCs for clinical disease treatment.
Disclosure of Invention
The invention aims to overcome the problems of difficult long-term storage and direct use in the prior art, and provides an application of trehalose and betaine in cryopreserved cells.
In order to achieve the above object, the present invention provides, in a first aspect, a cell preparation comprising cells, trehalose and betaine, wherein the weight ratio of trehalose to betaine is 1.
In a second aspect, the invention provides the use of trehalose and betaine to promote cell cryopreservation viability or improve cell transplantation safety.
In a third aspect, the present invention provides a method of cryopreserving cells, the method comprising: mixing the cells, trehalose and betaine in the presence of a buffer; then freezing and storing the obtained cell mixture; wherein the weight ratio of the trehalose to the betaine is 1-4.2.
Through the technical scheme, the cell culture medium can realize long-term preservation of cells (particularly mesenchymal stem cells), can be directly used without complicated steps such as recovery culture amplification and washing, saves the time for recovering the cultured cells and operation platforms (laminar flow chambers, superclean tables, cell culture boxes, pipettors, culture solutions and the like) required by cell operation, and is favorable for saving equipment and labor cost. In addition, the invention can avoid cell curative effect difference caused by inconsistent cell states and uneven cell quality due to operation of different operators. In vivo experiments prove that compared with a cryopreservation system of 10% dimethyl sulfoxide, the cell preparation disclosed by the invention has extremely strong advantages in clinical use safety. Therefore, the invention is particularly beneficial to break the predicament of the current mesenchymal stem cell clinical therapy.
Drawings
FIG. 1 shows the state of the cells recovered after freezing for 7 days in each experiment of example 1;
FIG. 2 shows the state of the cells recovered after freezing for 60 days in each experiment of example 2;
FIG. 3 shows the state of the cells recovered after freezing for 7 days in each experiment of example 3;
FIG. 4 is a graph of the change in the levels of mouse ALT and AST after cryopreservation of cell preparations obtained according to a preferred embodiment of the invention and after liver injury in mice treated with conventional MSCs transplantation;
FIG. 5 is a graph comparing in vivo safety tests for different substances.
Detailed Description
The endpoints of the ranges and any values disclosed herein are not limited to the precise range or value, and such ranges or values should be understood to encompass values close to those ranges or values. For ranges of values, between the endpoints of each of the ranges and the individual points, and between the individual points may be combined with each other to give one or more new ranges of values, and these ranges of values should be considered as specifically disclosed herein.
The invention provides a cell preparation, which is characterized by comprising cells, trehalose and betaine, wherein the weight ratio of the trehalose to the betaine is 1-4.2, for example, 1:1.2,1:1.8,1:1.9,1:2.2,1:3,1:3.5,1:3.8,1:3.9,1:4,1:4.1,1:4.2, or any range or value between the above values, preferably 1.
According to the invention, in relation to 10 7 -10 9 A cell (especially 10) 7 Individual cells), preferably in an amount of 10-100mg, such as 15, 18, 19, 20, 21, 22, 25, 28, 35, 45, 55, 65, 75, 85, 95mg or any range or value therebetween, more preferably 18-22mg.
According to the present invention, the cell preparation may be a liquid preparation and the content of cells may be 10 7 -10 9 Individual cells/mL.
According to the invention, the cell preparation may be a liquid preparation and the trehalose is preferably present in an amount of 18-22mg/mL, such as 18, 18.5, 19, 19.5, 19.8, 20, 20.2, 20.5, 21, 21.5, 22mg/mL or any range or value between the above values.
According to the invention, the cell preparation may be a liquid preparation and the betaine content is preferably 78-82mg/mL, such as 78, 78.5, 79, 79.5, 79.8, 80, 80.2, 80.5, 81, 81.5, 82mg/mL or any range or value between the above values.
According to the present invention, in order to prepare a liquid formulation, physiological saline may be used, and thus the liquid formulation may further contain physiological saline.
According to a preferred embodiment of the invention, the cell preparation consists of cells only, physiological saline, trehalose and betaine.
The inventor of the present invention finds that trehalose and betaine are particularly suitable for cryopreservation of mesenchymal stem cells, and compared with common cell cryopreservation protective agents such as glycerol and dimethyl sulfoxide, trehalose and betaine are more advantageous in cryopreservation of cells, and have higher cell cryopreservation activity, longer preservation time and higher safety, so that preferably, the cells are mesenchymal stem cells, and more preferably at least one of adipose mesenchymal stem cells, bone marrow mesenchymal stem cells, umbilical cord blood mesenchymal stem cells and dental pulp mesenchymal stem cells.
The invention also provides application of the trehalose and the betaine in promoting cell cryopreservation activity or improving cell transplantation safety.
The invention also provides a method for cryopreserving cells, which is characterized by comprising the following steps: mixing the cells, trehalose and betaine in the presence of a buffer; then freezing and storing the obtained cell mixture; wherein the weight ratio of trehalose to betaine is as described above and will not be described in detail.
In the present invention, the obtained cell mixture may be frozen by a conventional freezing method, but the inventors of the present invention found that freezing in a specific manner further improves the cell freezing viability and prolongs the storage time, and therefore, in a preferred embodiment of the present invention, the method of freezing the obtained cell mixture includes: sequentially placing the obtained cell mixture at-5 ℃ to-3 ℃ for 8-15 minutes; 25-35 minutes at-25 ℃ to-15 ℃; 15-20 hours at-85 ℃ to-75 ℃; -200 ℃ to-190 ℃ liquid nitrogen preservation, most preferably, the manner of subjecting the resulting cell mixture to cryopreservation comprises: sequentially placing the obtained cell mixture at-5 ℃ to-3 ℃ for 9-11 minutes; 29-31 minutes at-21 ℃ to-19 ℃; 16-18 hours at-81 ℃ to-79 ℃; preserving with liquid nitrogen at-197 deg.C to-195 deg.C.
The present invention will be described in detail below by way of examples. In the following examples, the mesenchymal stem cells are derived from the stem cell engineering technology center of shanxi province and are canine adipose mesenchymal stem cells; d-trehalose was purchased from Shandong Yousio chemical science and technology, inc.; betaine was purchased from Shandong Yousio chemical science and technology, inc.; the viability detection method of the mesenchymal stem cells refers to a Giemsa staining result after cell recovery, and ImageJ software is used for quantitatively analyzing the cell number, wherein the viability is = the cell number recovered after cryopreservation/the cell number before cryopreservation multiplied by 100%, and the results are averaged.
Example 1
(1) Resuscitating mesenchymal stem cells, culturing with 10mL/100mL serum replacement-added alpha-MEM
Based on 37 ℃,5% CO 2 Normally culturing and subculturing in an incubator;
(2) digesting the adherent culture 4 th generation mesenchymal stem cells, detecting the activity of the adherent culture 4 th generation mesenchymal stem cells, and counting;
(3) will be 1 × 10 7 The mesenchymal stem cells were transferred to a 1.5mL centrifuge tube and centrifuged at 1500rpm/min for three minutes.
Discarding supernatant, precipitating to obtain mesenchymal stem cells, and preparing 64 groups of cells in total, wherein each group comprises 3 cells in parallel;
(4) adding 0.9% physiological saline, betaine stock solution (betaine concentration is 1mg/μ L) and D-trehalose stock solution (D-trehalose concentration is 1mg/μ L), mixing, and adding volume of each component
As shown in table 1;
(5) the cell preparation obtained by mixing the above components uniformly is preserved by programmed freezing (10 min at 4 ℃;
30 minutes at-20 ℃; -80 ℃ for about 17 hours; liquid nitrogen storage at-196 ℃).
(6) After being preserved for 7 days by liquid nitrogen, the cell preparation is put into water bath at 37 ℃ for heating for 2min, the activity of the mesenchymal stem cells and the like are detected, the cell states recovered after being preserved for 7 days in each group of experiments are shown in figure 1, and the activity detection results are shown in table 1.
(7) The cell preparation after the preparation of the group 34 is heated in water bath is used for treating the mice with acute liver injury. Selecting 6-8 week old Kunming white mice 20, and averagely dividing into 4 groups (NC group, CCl) 4 Group, MSC-P group), 2. Mu.L/g CCl was used 4 Dissolving in olive oil, and injecting into abdominal cavity of the mice of the latter three groups, and injecting into abdominal cavity of the NC group with the same amount of olive oil. After 2 hours of intraperitoneal injection, MSC group mice were normally cultured by tail vein transplantation with non-cryopreserved MSC 2X 10 6 The cell preparation after heating in water bath of group 34 of example 1 was transplanted from tail vein to each mouse in MSC-P group. Mouse serum was collected 24 hours later and analyzed for mouse serum alanine Aminotransferase (ALT) and aspartate Aminotransferase (AST) levels. The results are shown in FIG. 4, where MSC-P treated in group 34 of example 1 has similar therapeutic effects including recovery effects on ALT and AST in mice compared to MSC transplanted immediately after normal culture without freezing and with addition of only 99% physiological saline.
TABLE 1
Example 2
Cryopreservation was performed as in groups 34 and 64 of example 1, except that the cryopreservation time was 60 days, the cell preparation was placed in a 37 ℃ water bath and heated for 2min, and the viability of the mesenchymal stem cells was measured, and the results were 95.2% and 100.7%, respectively, and the cell state was as shown in fig. 2.
Example 3
Cryopreservation was carried out as in groups 34 and 64 of example 1 except that the conditions for freezing in step (5) were such that the preparation was directly transferred to liquid nitrogen at-196 ℃ for storage, and the viability assay gave 80% and 83%, respectively, and the cell status is shown in FIG. 3.
Example 4
30 Kunming white mice, 6-8 weeks old, were selected and divided on average into 10 groups (see Table 2, NC groups, 10% betaine, 50% betaine, 100% betaine, 10% trehalose, 50% trehalose, 100% trehalose, 10% DMSO; 50% DMSO; 100% DMSO). Serum was collected from the mice 2 hours after intraperitoneal injection to analyze the levels of alanine Aminotransferase (ALT), aspartate Aminotransferase (AST), creatinine (Cr) and urea nitrogen (BUN) in the serum. The results are shown in fig. 5, in which the indexes of the mice in groups 1-7 are all in the normal range, and the damage degree of the mice in groups 8-10 increases with the increase of the concentration of DMSO due to different degrees of damage to liver and kidney caused by DMSO, thus, the cell preparation of the present invention is obviously superior in clinical safety.
TABLE 2
| Group of | Physiological saline (mu L) | Betaine (mu L) | D-trehalose (μ L) | Dimethyl sulfoxide (mu L) |
| 1 | 100 | 0 | 0 | 0 |
| 2 | 900 | 100 | 0 | 0 |
| 3 | 500 | 500 | 0 | 0 |
| 4 | 0 | 1000 | 0 | 0 |
| 5 | 900 | 0 | 100 | 0 |
| 6 | 500 | 0 | 500 | 0 |
| 7 | 0 | 0 | 1000 | 0 |
| 8 | 900 | 0 | 0 | 100 |
| 9 | 500 | 0 | 0 | 500 |
| 10 | 0 | 0 | 0 | 1000 |
The preferred embodiments of the present invention have been described above in detail, but the present invention is not limited thereto. Within the scope of the technical idea of the invention, many simple modifications can be made to the technical solution of the invention, including various technical features being combined in any other suitable way, and these simple modifications and combinations should also be regarded as the disclosure of the invention, and all fall within the scope of the invention.
Claims (10)
1. A cell preparation, which is characterized by comprising cells, trehalose and betaine, wherein the weight ratio of the trehalose to the betaine is 1.
2. The cell preparation of claim 1, wherein the weight ratio of trehalose to betaine is 1.
3. The cell preparation of claim 1 or 2, wherein relative to 10 7 -10 9 The content of the trehalose is 10-100mg per cell.
4. The cell preparation of claim 1 or 2, wherein relative to 10 7 -10 9 The content of the trehalose in the cells is 18-22mg.
5. The cell preparation according to claim 1 or 2, wherein the cell preparation is a liquid preparation and the content of cells is 10 7 -10 9 Individual cells/mL;
or the cell preparation is a liquid preparation and the content of the trehalose is 18-22mg/mL;
or the cell preparation is a liquid preparation and the content of the betaine is 78-82mg/mL.
6. The cell preparation of claim 5, wherein the liquid preparation further comprises a physiological saline.
7. The cell preparation of claim 1, wherein the cells are mesenchymal stem cells, preferably at least one of adipose mesenchymal stem cells, bone marrow mesenchymal stem cells, umbilical cord blood mesenchymal stem cells, dental pulp mesenchymal stem cells.
8. The application of trehalose and betaine in promoting cell cryopreservation activity or improving cell transplantation safety.
9. A method of cryopreserving cells, the method comprising: mixing the cells, trehalose and betaine in the presence of a buffer; then freezing and storing the obtained cell mixture; wherein the weight ratio of the trehalose to the betaine is 1-4.2.
10. The method of claim 1, wherein the weight ratio of trehalose to betaine is 1.
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Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5827640A (en) * | 1996-06-14 | 1998-10-27 | Biostore New Zealand Limited | Methods for the preservation of cells and tissues using trimethylamine oxide or betaine with raffinose or trehalose |
| US20020177116A1 (en) * | 1996-06-14 | 2002-11-28 | Biostore New Zealand Ltd. | Compositions and methods for the preservation of living tissues |
| US20060257842A1 (en) * | 2003-05-29 | 2006-11-16 | Pettegrew Jay W | Cryopreservation media and molecules |
| CN105263320A (en) * | 2013-06-12 | 2016-01-20 | 那慕尔大学 | Freeze preservation of living cells |
| CN106234352A (en) * | 2016-08-02 | 2016-12-21 | 中国农业科学院特产研究所 | A kind of reduce the method for organelle damage after freezen protective oocyte freeze thawing |
| CN108434105A (en) * | 2017-02-16 | 2018-08-24 | 华东理工大学 | Vaccine preparation method and preparation based on the accumulation of intracellular glycine betaine |
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- 2022-12-19 CN CN202211631298.7A patent/CN115868478B/en active Active
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5827640A (en) * | 1996-06-14 | 1998-10-27 | Biostore New Zealand Limited | Methods for the preservation of cells and tissues using trimethylamine oxide or betaine with raffinose or trehalose |
| US20020177116A1 (en) * | 1996-06-14 | 2002-11-28 | Biostore New Zealand Ltd. | Compositions and methods for the preservation of living tissues |
| US20060257842A1 (en) * | 2003-05-29 | 2006-11-16 | Pettegrew Jay W | Cryopreservation media and molecules |
| CN105263320A (en) * | 2013-06-12 | 2016-01-20 | 那慕尔大学 | Freeze preservation of living cells |
| CN106234352A (en) * | 2016-08-02 | 2016-12-21 | 中国农业科学院特产研究所 | A kind of reduce the method for organelle damage after freezen protective oocyte freeze thawing |
| CN108434105A (en) * | 2017-02-16 | 2018-08-24 | 华东理工大学 | Vaccine preparation method and preparation based on the accumulation of intracellular glycine betaine |
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